This project is oriented towards determining factors that regulate the fate of transmembrane proteins both on the cell surface and in the secretory pathway. There is evidence from yeast that a ubiquitin protein ligase (Rsp5) plays a significant role in the internalization and subsequent degradation in vacuoles of several multi-membrane spanning transporters and receptors. We have cloned a murine homolog of Rsp5 (Nedd4) and more recently obtained cDNAs encoding others. For mammalian tyrosine kinase containing and tyrosine kinase coupled receptors members of the Cbl family of ubiquitin ligases play important roles in targeting of receptors for degradation. Cbl family proteins bind to these receptors resulting in the targeting for degradation of the receptors, the associated Cbl family proteins and other components of the signaling pathway. We have now established that members of the Cbl family are themselves constitutively targeted for degradation through interaction with Nedd4 and related HECT domain E3s. Expression of catalytically active Nedd4 therefore reverses the effect of Cbl family proteins in the down regulation of EGFR. This targeting is dependent on the catalytic activity of the HECT domain E3s but independent of the E3 activity of Cbl proteins. These results provide the first example of one class of ubiquitin ligase regulating the activity of another. Studies are currently underway both to map the sites of interaction between Cbl and Nedd4 family proteins and to determine whether dysregulation of Nedd4 family expression might play a role in aberrantly enhanced signaling in cancer. Other aspects of this project are oriented toward determining factors that influence the fate of proteins along the secretory pathway. We have now established that degradation of two subunits of the T cell antigen receptor from the ER involves ubiquitination and targeting to proteasomes. Inhibition of proteasome function results in the accumulation of these subunits in membrane-bound forms. In yeast, degradation from the ER has been shown to involve two enzymes of the ubiquitin conjugating system known as UBC6 and UBC7. UBC6 is a type-IV membrane anchored protein while UBC7 has no membrane anchor. We have isolated mammalian homologs of both UBC6 and UBC7 and have determined that they are localized to the ER membrane and that one of these, the murine homolog of UBC7 (MmUBC7) plays a role in targeting the T cell antigen receptor subunits for degradation from the ER. We have subsequently determined that UBC7 specifically interacts with a RING finger protein known as gp78, also known as the autocrine motility factor receptor, and that this receptor is capable of targeting unassembled TCR subunits as well as other proteins for degradation from the ER. We are now in the process of analyzing the structure of gp78. To date we have determined that this E3 uniquely binds MmUBC7/Ube2g2 through a region outside of the RING finger. This region has been established to be less than 30 amino acids and crucial regions within this domain have now been identified. The potential for this sequence to be employed towards the development of clinical tools has resulted in the filing of a patent. We have also established direct binding of both mono-ubiquitin and polyubiquitin chains to the Cue domain of gp78. We have determined that the RING finger, Cue domain and E2 binding site are all required for gp78 to target both itself and heterologous substrates for degradation. gp78 levels are correlated with the metastatic potential of tumors including melanomas and lung cancers.